Protein–Ligand Interaction Analyses with Nuclear Magnetic Resonance Spectroscopy Enhanced by Dissolution Triplet Dynamic Nuclear Polarization
Koichiro Miyanishi, Toshihiko Sugiki, Takafumi Matsui, Ryotaro Ozawa, Y. Hatanaka, Hideo Enozawa, Yushi Nakamura, T. Murata, Akinori Kagawa, Yasushi Morita, Toshimichi Fujiwara, Masahiro Kitagawa, Makoto Negoro
Abstract
Solution-state nuclear magnetic resonance spectroscopy (NMR) is a powerful method for the analysis of intermolecular interactions within a biomolecular system. However, low sensitivity is one of the major obstacles of NMR. We improved the sensitivity of solution-state 13 C NMR for the observation of intermolecular interactions between protein and ligand using hyperpolarized solution samples at room temperature. Eutectic crystals composed of 13 C-salicylic acid and benzoic acid doped with pentacene were hyperpolarized by dynamic nuclear polarization using photoexcited triplet electrons, and a 13 C nuclear polarization of 0.72 ± 0.07% was achieved after dissolution. The binding of human serum albumin and 13 C-salicylate was observed with several hundred times sensitivity enhancement under mild conditions. The established 13 C NMR was applied for pharmaceutical NMR experiments by observation of the partial return of the 13 C chemical shift of salicylate by competitive binding with other non-isotope-labeled drugs.